In the tissue, the cells are embedded in the native extracellular matrix (ECM), which is an important part of the cellular environment. The native ECM is a highly ordered, tissue-specific network which consists of collagens, glycoproteins, proteoglycans and glycosaminoglycans (GAG). The composition for various tissue and for various stages of development is very different here, such that the respective matrix has specific properties with respect to interaction with cells and growth factors.
The main structural protein of the native bone matrix is collagen type I, but various other matrix proteins such as proteoglycans and glycoproteins can interact with the collagen and influence the structure and function of the matrix. These noncollagenic ECM proteins fulfill specific functions in the matrix. Thus fibronectin, in addition to cell-binding properties, also has collagen- and GAG-binding properties [Stamatoglou and Keller, 1984, Biochim Biophys Acta. Oct 28; 719(1): 90-7], whereas small leucine-rich proteins (SLRPs) such as decorin not only play a role in the organization of the native ECM (decorin modulates fibrilogenesis in vivo), but also bind growth factors such as TGF-β or even play a role as signal molecules [Kresse and Schönherr, 2001, J Cell Phys 189: 266-274].
Proteoglycans and glycoproteins differ by their degree of glycosylation, the sugar content of the particularly highly glycosylated proteoglycans consisting of various glycosaminoglycans. The distribution of these chains can be tissue-specific, as, for example, for decorin (chondroitin sulfate in the bone, dermatan sulfate in the skin). The glycosaminoglycans are large, unbranched polysaccharides which consist of repeating disaccharides, which are composed, for example, of N-acetyl-galactosamine, N-acetylglucosamine, glucuronate or iduronate, which are sulfated to different degrees. The sugar chains are present in vivo bound to the proteoglycans and play an important role in the function of these proteins, i.e. in growth factor binding and modulation [Bernfield et al, 1999, Annu Rev Biochem, 68: 729-777].
Individual ECM constituents, in particular collagen, are already utilized for the biocompatible modification of scaffolds and implants in order to improve cell adhesion and tissue integration. In addition to collagen, further ECM components such as polysaccharides are used in various applications. Thus bone tissue was crosslinked with glycosaminoglycans in order to produce a three-dimensional scaffold for applications in tissue culture (WO 01/02030A2).
A chondroitin sulfate-containing mixture is used for the repair of bone defects; this promotes the healing of the connective tissue, mainly on account of the content of aminosugars and increased matrix production caused thereby (WO 98/27988, WO 99/39757). In combination with collagen, plant polysaccharides are used as wound coverings (EP 0140569 A2), and a combination of chitosan and GAGs is described as an agent for the stimulation of the regeneration of hard tissue (WO 96/02259). Collagen-GAG mixtures are produced here by acid coprecipitation, an unstructured precipitate and no defined collagen fibrils comparable to those in the native ECM being formed (U.S. Pat. Nos. 4,448,718, 5,716,411, 6,340,369).
With progressive availability of recombinant growth factors, those osteoinductive factors which actively influence the interactions between implants and surrounding tissue are increasingly of interest for implant applications [Anselme K (2000). Biomaterials 21, 667-68]. In connection with bone healing, the ‘bone morphogenetic proteins’ (BMP 2, 4-7) are particularly interesting since they induce the differentiation of mesenchymal stem cells in chondrocytes and osteoblasts and the formation of new bone [Celeste A J, Taylor R, Yamaji N, Wang J, Ross J, Wozney J M (1994) J. Cell Biochem. 16F, 100; Wozney J M, Rosen (1993). Bone morphogenetic proteins in Mundy, G R, Martin T J (Ed.) Physiology and pharmacology off bone. Handbook of experimental pharmacology, Vol. 107. Springer Verlag, Berlin, 725-748]. On account of these strong bone-inducing effects, recombinant BMPs are employed in various carrier materials in order to promote and to improve the regeneration of bone. Effective carriers for morphogenetic proteins should bind these, protect against hydrolysis, make possible subsequent, controlled release and promote the associated cell reactions. Moreover, such carriers should be biocompatible and biodegradable. Preferred carrier materials for BMPs are, for example, xenogenic bone matrix (WO 99/39757) or natural tissue subsequently crosslinked with GAGs (WO 01/02030 A2), or HAP, collagen, TCP, methylcellulose, PLA, PGA, and various copolymers (EP 0309241 A2, DE 19890329, EP 0309241 A2, DE 19890906, WO 8904646 A1, DE 19890601). Further applications comprise a crosslinked synthetic polymer which can contain additional components such as GAGs, collagen or bioactive factors (WO 97/22371), or crosslinked collagen mixed with glycosaminoglycans and osteogenic factors (WO 91/18558, WO 97/21447). The collagen-GAG mixture is in this case likewise produced by acid coprecipitation.
The use of recombinant growth factors is associated with great disadvantages. Since the recombinant factors usually have a lower activity than the endogenous factors occurring naturally in the tissue, in order to achieve an effect in vivo unphysiologically high doses are necessary. The administration of recombinant factors can only simulate the action of endogenous factors very incompletely.
By the use of factors which promote the action of the BMPs (Bone morphogenetic protein), or by the use of cells which can express the growth factors in situ, it is attempted to minimize or to circumvent this problem (WO 97/21447, WO 98/25460). Further problems can result from the fact that receptors for BMP occur in many different tissues; the function of these growth factors is thus not limited to the bone.